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Transgenic Research
Associated with the International
Society for Transgenic Technologies
(ISTT)
ISSN 0962-8819
Volume 23
Number 2
Transgenic Res (2014) 23:377-388
DOI 10.1007/s11248-013-9759-7
Development of transgenic sweet potato
with multiple virus resistance in South
Africa (SA)
B.J.Sivparsad & A.Gubba
1 23
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ORIGINAL PAPER
Development of transgenic sweet potato with multiple virus
resistance in South Africa (SA)
B. J. Sivparsad
•
A. Gubba
Received: 18 January 2013 / Accepted: 3 October 2013 / Published online: 25 October 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Multiple infections of Sweet potato feath-
ery mottle virus (SPFMV), Sweet potato chlorotic
stunt virus (SPCSV), Sweet potato virus G (SPVG)
and Sweet potato mild mottle virus (SPMMV) cause a
devastating synergistic disease complex of sweet
potato (Ipomoea batatas Lam.) in KwaZulu-Natal,
South Africa. In order to address the problem of
multiple virus infections and synergism, this study
aimed to develop transgenic sweet potato (cv. Bles-
bok) plants with broad virus resistance. Coat protein
gene segments of SPFMV, SPCSV, SPVG and
SPMMV were used to induce gene silencing in
transgenic sweet potato. Transformation of apical tips
of sweet potato cv. Blesbok was achieved by using
Agrobacterium tumefaciens strain LBA4404 harbor-
ing the expression cassette. Polymerase chain reaction
and Southern blot analyses showed integration of the
transgenes occurred in six of the 24 putative transgenic
plants and that all plants seemed to correspond to the
same transformation event. The six transgenic plants
were challenged by graft inoculation with SPFMV,
SPCSV, SPVG and SPMMV-infected Ipomoea setosa
Ker. Although virus presence was detected using
nitrocellulose enzyme-linked immunosorbent assay,
all transgenic plants displayed delayed and milder
symptoms of chlorosis and mottling of lower leaves
when compared to the untransformed control plants.
These results warrant further investigation on resis-
tance to virus infection under field conditions.
Keywords Genetic engineering Sweet potato
viruses Transgenic sweet potato
Virus resistance
Introduction
Sweet potato (Ipomoea batatas Lam.) is an important
food staple for poor and rural households in South
Africa (SA). The crop is favoured by resource-poor
farmers due to its good performance under adverse
farming conditions, peak productivity in small farm-
ing areas and high carbohydrate and vitamin content
(Naylor et al. 2004). However, the common practice of
cutting and propagating vines in newly planted fields
has resulted in the rampant spread and build-up of
virus disease which has subsequently led to a
discernable decline in tuber yield and/or quality over
time (Clark et al. 2002; Domola et al. 2008). As a
result, the incidence of viral disease in major
Electronic supplementary material The online version of
this article (doi:10.1007/s11248-013-9759-7) contains supple-
mentary material, which is available to authorized users.
B. J. Sivparsad (&) A. Gubba
Department of Plant Pathology, School of Agricultural,
Earth and Environmental Sciences, University of
KwaZulu-Natal, Private Bag X01, Scottsville,
Pietermaritzburg 3209, South Africa
e-mail: 201297209@stu.ukzn.ac.za
A. Gubba
e-mail: gubbaa@ukzn.ac.za
123
Transgenic Res (2014) 23:377–388
DOI 10.1007/s11248-013-9759-7
Author's personal copy
sweetpotato growing provinces in SA has hindered the
successful cultivation and production of this important
crop. The problem is exacerbated by the frequent
occurrence of multiple viruses infecting sweet potato
(Domola et al. 2008).
Infection of sweet potato by multiple viruses is a
common occurrence and often results in a viral
synergism which appears as a more severe disease
than the sum effect of infection with each virus alone
(Mukasa et al. 2006; Untiveros et al. 2007). The most
devastating viral disease affecting sweet potatoes
worldwide is sweet potato virus disease (SPVD)
(Kokkinos et al. 2006; Miano et al. 2008) and is
caused by the synergistic interaction between Sweet
potato feathery mottle virus (SPFMV) and Sweet
potato chlorotic stunt virus (SPCSV). SPFMV, a
member of the genus Potyvirus in the family Potyvir-
idae, is the most common sweet potato virus, found
nearly everywhere sweet potatoes are grown (Moyer
and Salazar 1989; Karyeija et al. 1998; Kreuze and
Fuentes 2008). SPCSV, a member of the genus
Crinivirus in the family Closteroviridae, is one of
the most devastating viruses infecting sweet potato
worldwide (Winter et al. 1992; Mukasa et al. 2006;
Untiveros et al. 2007) due to its ability to break down
the natural resistance of sweet potato to other viruses
and mediate severe synergistic diseases with other
sweet potato viruses (Karyeija et al. 2000). The most
common and severe of these diseases is SPVD which
is caused by co-infection with SPFMV (Kreuze 2002).
Sweet potato plants infected with SPVD display
chlorosis, small deformed leaves, severe stunting and
an almost 99 % reduction in tuber yield (Gibson et al.
1998; Karyeija et al. 1998; Tairo et al. 2005). The
occurrence of SPVD and other viral co-infections has
been reported in almost every sweet potato growing
area (Karyeija et al. 1998; Loebenstein et al. 2009). It
has been hypothesized that other yet unidentified
viruses are involved in SPVD and enhance replication
of the potyviruses. Efforts to identify unknown viruses
in the United States have revealed the presence of
Tomato spotted wilt virus (TSWV) in sweet potato
(Clark and Hoy 2007). It is still not known how
widespread this virus is or what effect it has on yield
(Valverde et al. 2007). A recent survey for sweet
potato-infecting viruses demonstrated that Sweet
potato virus G (SPVG) and Sweet potato mild mottle
virus (SPMMV) are also able to form synergistic
associations with SPCSV and SPFMV to form a new
SPVD complex that is commonly detected in sweet
potato grown in KwaZulu-Natal (KZN) in SA (Siv-
parsad 2013).
The frequent occurrence of mixed synergistic viral
complexes has complicated efforts to control viral
diseases in sweet potato. Currently, no single man-
agement tool is available to provide adequate control
of viral complexes that infect sweet potato. The use of
healthy planting material, phytosanitation and cultural
measures have been used with minimal success due to
difficulties encountered when trying to integrate these
strategies into the subsistence production systems
practiced by resource-poor farmers (Gibson et al.
2004; Nyaboga et al. 2008). Little success has also
been reported in the breeding for virus resistance in
sweet potato cultivars (Gibson et al. 1998, 2004;
Karuri et al. 2009). Factors such as virus variation,
time and expenditure required has mired conventional
breeding efforts (Lomonossoff 1995). Moreover,
genetic sources of resistance are scarce and the
incorporation of such resistance from the wild diploid
species into polyploid sweet potato is a complicated
task (Kreuze 2002).
Alternate strategies for obtaining virus resistance
through biotechnology-based strategies have the
potential to serve as valuable interventions in sweet
potato virus control (Kreuze 2002). Most of these
strategies are based on the concept of ‘pathogen-
derived resistance’ (PDR), which proposes that path-
ogen resistance genes may be developed from the
pathogen’s own genetic material (Sanford and John-
ston 1985).
Genetic engineering efforts have been used to
develop transgenic sweet potato with virus resistance
using the coat protein gene of SPFMV and/or SPCSV
(Newell et al. 1995; Gama et al. 1996; Otani et al.
1998; Okada et al. 2002; Kreuze et al. 2008).
However, given the distribution and genetic diversity
of sweet potato viruses under field conditions this
approach has had limited success. Other strategies to
generate transgenic sweet potato with resistance to
SPVD have also been tested. Rice cysteine proteinase
inhibitor (OCI) mediated resistance to potyviruses is
believed to inhibit the viral cysteine proteinase NIa
that processes the potyviral polyprotein (Gutie
´
rrez-
Campos et al. 1999). The expression of OCI in
transgenic plants might also confer resistance to
SPCSV, as closteroviruses also encode cysteine pro-
teinases to modify some of their proteins. Sweet potato
378 Transgenic Res (2014) 23:377–388
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plants transformed with the OCI has been reported to
show increased resistance to SPFMV (Cipriani et al.
2001). However, the resistance was not effective and
typical symptoms of SPVD developed in plants
infected with SPCSV and SPFMV (Clark et al. 2012).
Previous studies have indicated that transgenes
consisting of 110–218 bp nucleocapsid (N) gene seg-
ments of TSWV linked to the 720-bp green fluorescent
protein (GFP) gene confer resistance to TSWV but not
of 24–59 bp N gene segments of TSWV similarly
linked to GFP (Jan et al. 2000a; Pang et al. 1997). Also,
a single chimeric gene consisting of linked viral
segments of TSWV and Turnip mosaic virus (TuMV)
confers resistance to both viruses (Jan et al. 2000b).
Recently, Lin et al. (2011) demonstrated that dual
virus resistance is achieved by fusing the partial C2
gene of Tomato leaf curl Taiwan virus (ToLTWCV) to
the middle half of the TSWV N gene. The study
demonstrated that both viruses (ToLCTWV and
TSWV) and foreign transgenes could induce gene
silencing. In addition, these studies suggested that
resistance can be induced by a transcribed DNA
segment (middle half of N gene) known as the ‘silencer
DNA’ and that this segment can also be used as a
transgene and render TSWV resistance to transgenic
plants.
This study aimed to develop transgenic sweet
potato (cv. Blesbok) with mutiple virus resistance.
Coat protein (CP) gene fragments of the four most
prevalent viruses infecting sweet potato in KZN were
fused and introduced into sweet potato via Agrobac-
terium tumefaciens-mediated transformation. Trans-
genic sweet potato plants showing varying levels of
resistance to SPFMV and SPVG were developed.
Materials and methods
Plant materials
Sweet potato cultivar ‘Blesbok’ was obtained from Dr.
P. Shanahan. The cultivar Blesbok, released in 1989 is
a popular cultivar in SA, accounting for 80 % of
national production with yields averaging 45 t/ha
(Laurie, 2002). Stock plants were tested for virus
infection using standard nitrocellulose membrane
enzyme-linked immunosorbent assay (NCM-ELISA
kits) obtained from the International Potato Centre
(CIP), Lima, Peru. Vine cuttings of virus-free plants
were potted in sterilized potting medium and main-
tained at 25 °C in an insect-proof greenhouse in the
Controlled Environment Research Unit (CERU) at the
University of KwaZulu-Natal (UKZN-PMB). These
plants were fertilized weekly with 3:1:3 (nitro-
gen:phosphate:potassium) and used as stock plants
for the establishment of virus-free in vitro plantlets,
according to the protocol described by Sivparsad and
Gubba (2012a). Stock plants were maintained in the
greenhouse for up to 1 year and were kept in highly
vegetative state by constant cutting back of stems.
Vector construction
Isolates of SPFMV, SPCSV, SPVG and SPMMV were
identified and isolated from rural farms in Umbumb-
ulu (Sivparsad 2013). A low genetic variability among
these isolates from KZN was previously determined
(Sivparsad and Gubba 2012b). The CP gene-encoding
regions of SPFMV, SPCSV, SPVG and SPMMV were
chosen as target genes in the design of a segmented
gene construct. To create the construct, individual
fragments of the CP genes of SPFMV, SPCSV, SPVG
and SPMMV isolates were amplified by RT-PCR with
specific primers containing restriction sites for Xbal,
Xhol, Sall, Smal, and Apal. Each primer pair was
flanked by unique restriction sites and the SPFMV-F
primer, used for the amplification of the first segment
in the construct, was designed to contain a stop codon
(TAA) to ensure the production of untranslatable
transgene transcripts. Details of the primers used in
this study are given in Table S1 (in electronic
supplementary material). The individual segments
were then digested with their flanking restriction
enzymes and sequentially ligated in the sense orien-
tation to the plant expression vector pEPJ86-m/2N
(Jan 1998; kindly provided by Dennis Gonsalves) that
had been digested with the same restriction enzymes.
The pEPJ86-m/2N vector is a derivative of the plant
expression vector pEPJ86 and has an expression
cassette that contains the Cauliflower mosaic virus
(CaMV) 35S enhancer, promoter and terminator
sequences, the 5
0
untranslated region of the CP gene
of Alfalfa mosaic virus (AIMV) and the middle half of
the nucleocapsid protein (N) gene of TSWV. The
entire expression cassette is flanked by Hindlll and
Kpnl restriction sites (Jan 1998). After confirming the
presence of the each segment in the plant expression
cassette of pEPJ86-m/2N by polymerase chain
Transgenic Res (2014) 23:377–388 379
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reaction (PCR) using specific primers outlined (Table
S1 in electronic supplementary material), the resulting
plasmid was designated pEPJ86-m/2N-SPVD.
The expression cassette was digested out of pEPJ86-
m/2N-SPVD with HindIII and Kpnl and ligated into the
HindIII and Kpnl sites of the binary plant transforma-
tion vector pGA482G (Jan 1998; also kindly provided
by Dennis Gonsalves) that had been digested with the
same enzymes. The resulting plasmid was designated
pGA482G-SPVD (Fig. S1 in electronic supplementary
material). The pGA482G vector is a derivative of the
plant transformation vector pGA482 described by An
et al. (1988).
Polymerase chain reaction was performed to verify
the integration of each segment in pGA482G-SPVD.
Plasmid DNA of the pGA482G-SPVD was electropo-
rated into electrocompetent cells of the A. tumefaciens
strain LBA4404 using the MicroPulser
TM
Electropor-
ation Apparatus (BIO-RAD, RSA).
Plant transformation and regeneration
A single bacterial colony of A. tumefaciens strain
LBA4404 harboring the binary vector pGA482G-
SPVD was used to inoculate 5 ml of LB broth that
contained 50 mg/L rifampicin and 50 mg/L gentamy-
cin. The culture was grown in a shaking incubator
(220 rpm) at 28 °C until an optical density of 0.8–1
was reached at 600 nm. The culture was then trans-
ferred to 25 ml LB that contained 50 mg/L rifampicin,
50 mg/L gentamycin and 200 lM acetosyringone and
grown in an incubator with 220 rpm shaking at 28 °C
overnight. Bacterial cells of overnight cultures were
spun at 8,000 rpm for 20 min at 25 °C and resus-
pended in 25 ml of liquid Murashige and Skoog
(1962) medium (MS, Sigma-Aldrich, UK) containing
200 lM acetosyringone. Apical shoots (3-5 mm long)
with four to five primordia were excised from the
apical portions of 5 weeks old in vitro stock plants and
used as explants in transformation experiments.
Explants were submerged into the MS liquid suspen-
sion containing the A. tumefaciens strain LBA4404
harboring the binary vector pGA482G-SPVD and kept
in the dark at 28 ° C without agitation for 10 min. After
this co-culture step, explants were blotted onto sterile
filter paper and cultured in 9 cm Petri dishes contain-
ing 25 ml of shoot induction medium (SIM) consisting
of basic medium (BM) supplemented with 1 mg/L
BAP, 0.01 mg/L NAA, 50 mg/L kanamycin and
200 mg/L carbenicillin. The cultures were grown in
a growth room at 25 ± 2 °C under a 16 h photoperiod
with a light intensity of 54 lE/s/m
2
provided by white
fluorescent tubes. Cultures were transferred to new
medium every 3–4 weeks. Regeneration of putative
transgenic plants was achieved following organogen-
esis. Excess callus from regenerating shoots was
removed and elongating shoot tips were cultured in
glass culture vessels (Sigma-Aldrich, UK) containing
50 ml of root induction medium (RIM) supplemented
with 1 mg/L NAA and 25 mg/L kanamycin for
root development and plantlet maturation. After
2–4 weeks, plantlets were singly cultured in Magenta
GA7 vessels (Sigma-Aldrich, UK), each containing
100 ml of plant growth regulator (PGR) and antibi-
otic-free BM, for elongation of plantlets. The regen-
erated plantlets with fully developed roots were
transferred to 8 cm plastic pots filled with sterilized
seedling mix (Growmor, Cato Ridge), and placed in a
growth chamber at 25 ± 2 °C and 80 % relative
humidity (RH) under a 16 h photoperiod. After
2 weeks, plants were transferred to bigger pots
(20 cm) and placed in the greenhouse for
characterization.
Detection of transgenic events by PCR analysis
Total genomic DNA was isolated from leaf tissue of
putative transgenic and untransformed (control) plants
using the ZR Plant/Seed DNA Miniprep
TM
kit (Zymo
Research, SA). PCR analysis was used for initial
detection of putative transgenic plants. Primers (Table
S1 in electronic supplementary material) complemen-
tary to the individual transgene segments of SPFMV,
SPCSV, SPMMV and SPVG were used to detect the
integrity of the transgene construct in plants. In addition,
the primers NPTll-F (5
0
GATGCGCTGCGAATCG
GGAGCG 3
0
) and NPTII-R (5
0
GGAGAGGCTATTCG
GCTATGAC 3
0
) described by Yi et al. (2006) were used
to detect a 715 bp fragment of the integrated nptll gene.
Amplification was performed in an automated thermal
cycler programmed for one cycle of initial denaturation
of 95 °C for 2 min and 35 cycles of amplification with
1 min of denaturation at 95 °C, 1 min of annealing at
60 °C (SPFMV, SPCSV, SPVG, SPMMV) or 55 °C
(nptII) and 2 min of extension at 72 °C followed by one
cycle of final extension for 5 min at 72 °C.
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Detection of transgene integration by Southern
blot analysis
For Southern blot analysis, total plant DNA (20 lg)
from putative transgenic plants was digested overnight
at 37 °C with restriction endonuclease HindIII.
PGA482G-SPVD DNA digested with HindIII was
used as a positive control. In addition, undigested
genomic DNA from the T7 transgenic plant was also
included as a positive control to prove stable integra-
tion. A negative control consisted of untransformed
genomic plant DNA. Similarly, a second negative
control consisting of digested untransformed genomic
DNA spiked with HindIII-digested PGA482G DNA
was used to prove the authenicity of the transgene.
DNA fragments were separated by electrophoresis at
40 V for 3 h in a 1.2 % agarose gel and blotted onto
positively charged nylon membranes (Roche, Ger-
many) using the VacuGene XL Vacuum blotting
System (GE Healthcare Bio-Sciences, Sweden). DNA
fragments were fixed to the membrane by UV cross-
linking for 2–3 min (Stratalinker, Stratagene, CA,
USA). The hybridization probe corresponding to the
SPFMV segment was used for the detection of the
transgene and generated and labeled with alkali-labile
digoxigenin (DIG-dUTP) in PCR using the PCR DIG
Probe Synthesis kit (Roche, Germany). Hybridization
of the probe to membrane and subsequent chemilu-
minescence detection by enzyme immunoassay was
performed using the DIG High Prime DNA Labeling
and Detection Starter Kit II (Roche, Germany)
according to manufacturer’s instructions. Membranes
were exposed to Lumi-Film Chemiluminescent Detec-
tion Film (Roche, Germany) and film was developed
using standard procedures.
Greenhouse evaluation of transgenic plants
for virus resistance
Transgenic plants were assayed for virus resistance
according to the method described by Okada et al.
(2002). Vine samples that were NCM-ELISA positive
for infection with isolates of SPFMV, SPCSV, SPVG
and SPMMV were graft-inoculated onto 3 week-old
indicator plants, Ipomoea setosa Ker. using the crown-
cleft grafting. Two to three weeks post inoculation,
typical virus induced symptoms were observed on
growing leaves of indicator plants. The vine cuttings
from these virus-infected I. setosa plants served as
scions (inoculum) in grafting experiments. Five apical
cuttings from each of the six independently transformed
transgenic plants and two control plants were rooted
individually into pots containing sterilized potting
medium. All plants were kept at 25 °Cinaninsect-
proof growth room in the Controlled Environment
Research Unit (CERU) at the University of KwaZulu-
Natal (UKZN-PMB), under 80 % relative humidity, and
fertilized weekly with 3:1:3 (nitrogen:phosphate:potas-
sium). Approximately 4 weeks after planting, trans-
genic plants and controls were graft-inoculated with
scions of virus-infected I. setosa.Plantsweremain-
tained in the growth room for a period of 12 weeks to
allow for viruses to translocate through the graft and
progress into the transgenic sweet potato plant (Usugi
et al. 1990). Twelve weeks post inoculation, scions from
inoculated transgenic and control plants were graft-
inoculated back onto 3 week-old I. setosa plants.
Inoculated transgenic plants and corresponding inocu-
lated indicator plants were monitored for symptom
development and assayed for SPFMV, SPCSV, SPVG
and SPMMV by NCM-ELISA (CIP, Peru). In the NCM-
ELISA, a composite sample from each plant to be tested
was made by combining 1 cm leaf discs taken from the
top, middle and bottom levels of the plant.
Results
Plant transformation and regeneration
Callus formation started after 3 weeks on those
explants that survived repeated subculture and resid-
ual contamination by A. tumefaciens. Shoots formed
on SIM after 1 month and were allowed to elongate for
a further 3 weeks before being transferred to RIM. The
first regenerative roots formed, appeared after
1.5 months on RIM and were transferred to BM
without antibiotics to allow development into mature
plantlets. The first fully developed plants were
obtained 4 months from the beginning of the trans-
formation procedure. Overall, the regeneration of
transformed plants was significantly slower due to the
presence of selective antibiotics. Starting with 300
explants, 24 putative transformed plants were regen-
erated in the presence of kanamycin. All 24 plants
rooted well and appeared normal in morphology and
development resulting in a regeneration efficiency of
*8%.
Transgenic Res (2014) 23:377–388 381
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Detection of transgenic events by PCR analysis
PCR analysis confirmed the presence of each segment
of the transgene in six of the 24 putative transgenic
plants (Fig. 1). The size of each amplified product was
consistent with that of the original inserts of the CP
genes from SPFMV (319 bp), SPCSV (266 bp),
SPVG (213 bp) and SPMMV (168 bp). In addition,
the expected band of 715 bp from the coding region of
the nptll gene was found in all six plants. No PCR
products were observed in non-transgenic control
plants.
Detection of transgene integration by Southern
blot analysis
Southern blot analysis was performed to confirm
transgene integration and the number of insertion loci
for the six putative transgenic plants. The results
revealed the presence of one major hybridization band
specific to the transgene segment in all transformed
plants. A minor upper band apparently resulting from
an incomplete HindIII digestion is also visible
(Fig. 2). No signal was detected in both negative
control reactions. An estimated 16,366 bp band was
observed for the positive plasmid control. Undigested
genomic DNA from a transformed plant yielded a
hybridization signal which confirmed stable integra-
tion of the transgene in the genome of the transgenic
plants. All six transgenic plants showed an identical
hybridization pattern and one insertion loci. This
identical profile indicated that all transgenic plants are
not independent transformation events and are prob-
ably clones of each other. The overall transformation
efficiency (transgenic events/agroinoculated explants)
was 0.3 %.
A
B
C
D
E
Fig. 1 Detection of transgenic events by polymerase chain
reaction (PCR) analysis of the (a) Sweet potato feathery mottle
virus (SPFMV), (b) Sweet potato chlorotic stunt virus (SPCSV),
(c) Sweet potato virus G (SPVG), (d) Sweet potato mild mottle
virus (SPMMV) coat protein (CP) gene segments and (e) nptll
gene in putative transgenic sweet potato (Ipomoea batatas
Lam.) plants. Lanes 1 and 14 contain the O’ GeneRuler
TM
100 bp DNA ladder in (a–d) and the O’ GeneRuler
TM
1 kb plus
DNA ladder in (e). Lanes 2–13 and 15–26 contain PCR products
from DNA extracted from the 24 putative transgenic plants.
Lanes 27–29 in (a, d, e) and Lanes 28–29 in (b) and Lane 29 in
(c) show the PCR products of pGA482G-SPVD DNA. Lane 27
in (b) and Lanes 27–28 in (c) are blank. Lane 30 contains the
PCR product from DNA extracted from untransformed control
plants. Lane 31 shows the 323 bp product of the PCR positive
control reaction
382 Transgenic Res (2014) 23:377–388
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Greenhouse evaluation of transgenic plants
for virus resistance
At 10 weeks post inoculation, all non-transgenic
control plants displayed mild chlorosis and mosaic
whilst all transgenic plants remained symptomless.
Two weeks later, symptoms on non-transgenic control
plants progressed into severe mosaic, mottle, leaf
strap, vein clearing, crinkling and leaf curl (Fig. 3b/c).
In comparison to the control plants, all transgenic
plants displayed some degree of resistance in the form
of delayed and attenuated symptoms of chlorosis and
mottle (Fig. 3b/d). Transgenic plants T7 and T17
displayed a milder level of disease (mild mosaic)
compared to transgenic plants T1, T2, T3, and T4
(Table 1; Fig. 3a). The milder symptoms were mainly
observed in lower leaves, whilst many newly emerged
leaves remained symptomless. This suggested that the
spread of viruses from initially infected leaves may
have been inhibited.
The presence of SPFMV and SPVG was confirmed
by NCM-ELISA in all transgenic, non-transgenic plants
as well as their corresponding graft-inoculated I. setosa
plants (Table 1). Reactions of non-transgenic control
plants and the corresponding graft-inoculated I. setosa
were strong and indicated a high level of virus
accumulation in both the control and indicator plants.
Transgenic plants T1, T2, T3, and T4 displayed a weak
positive reaction whilst their corresponding graft-inoc-
ulated I. setosa displayed stronger positive reactions.
This would indicate a low level of virus accumulation in
these transgenic plants and a subsequent build up of
virus titer in indicator plants. Reactions of transgenic
plants T7 and T17 and their corresponding graft-
inoculated I. setosa plants were weak, indicating a low
level of virus accumulation in both the transgenic and
indicator plants. The presence of SPMMV and SPCSV
was not detected by NCM-ELISA in any of the
transgenic, non-transgenic plants and corresponding
graft-inoculated I. setosa plants. Therefore, graft-inoc-
ulation with SPMMV and SPCSV was not successful.
Discussion
Transgenic sweet potato plants of the cultivar Blesbok
showing resistance to multiple viruses were developed
in this study. The approach used in this study extends
from studies by Jan et al. (2000a, b) who demonstrated
that transgenic plants with resistance to multiple viruses
can be obtained by using a transgene construct consist-
ing of fused gene segments of different viruses. In this
study, a single segmented transgene construct contain-
ing four partial CP encoding gene segments of SPFMV,
SPCSV, SPVG and SPMMV fused to the middle half of
the N gene of TSWV. When originally described, the
role of the DNA silencer was to ensure that the length of
the transgene was long enough to induce gene silencing.
In certain cases, the DNA silencer itself can act as a
transgene and impart resistance to a virus as demon-
strated by Lin et al. (2011). However, since TSWV has
not been shown to infect sweet potato in SA, the DNA
silencer used in this study may not have a biological
effect on the transgenic plants developed.
Results indicated that resistance to SPFMV,
SPCSV, SPVG and SPMMV manifested as attenuated
and delayed symptom expression with possible
reduced virus titers. This is the first report of transgenic
sweet potato showing resistance to at least two viruses
of the SPVD complex.
The sweet potato transformation protocol reported
in this study resulted in a low efficiency. The use of
kanamycin in the selection of putative transformants
resulted in the regeneration of 24 plants out of 300
explants (*8 % regeneration efficiency). Subsequent
M N1 N2 P1 P2 1 2 3 4 7 17
Transgenic plants
23130bp---
9416bp---
6557b
p---
4361bp---
2322bp---
2027bp---
Fig. 2 Southern blot analyses of HindIII-digested total DNA
from transgenic sweet potato (Ipomoea batatas Lam.) plants
hybridized with a probe corresponding to the Sweet potato
feathery mottle virus (SPFMV) gene segment of the transgene.
The arrow indicates the hybridization signal of transgenic
plants. M = DIG labeled DNA molecular weight marker;
N1 = untransformed non-transgenic genomic plant DNA;
N2 = digested untransformed genomic DNA spiked with
HindIII-digested PGA482G DNA; P1 = digested PGA482G-
SPVD DNA (*16,366 bp); P2 = undigested genomic DNA
from a transgenic plant; 1, 2, 3, 4, 7, 17 = transgenic sweet
potato plants of cultivar Blesbok
Transgenic Res (2014) 23:377–388 383
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screening of putative regenerated transformants using
PCR (Fig. 1) and Southern blot analysis (Fig. 2)
confirmed transformation of 6 out of the 24 regener-
ated plants. It is possible that the low efficiency of
kanamycin in the selection of transformants likely led
to the low transformation efficiency reported in this
study. Low numbers of transgenic events of sweet
potato have also been reported in many previous
studies (Newell et al. 1995; Gama et al. 1996; Mora
´
n
et al. 1998; Cipriani et al. 2001; Otani et al. 2003; Song
et al. 2004; Kreuze et al. 2008). In addition, studies
have also reported the escape of untransformed
plantlets from selection using kanamycin (Luo et al.
2006; Xing et al. 2008).
B
C
D
non-transgenic transgenic
non-transgenic
transgenic
CONTROL T1 T2 T3
T4 T7 T17
A
Fig. 3 Greenhouse
evaluation of transgenic
sweet potato (Ipomoea
batatas Lam.) for resistance
against Sweet potato
feathery mottle virus
(SPFMV), Sweet potato
chlorotic stunt virus
(SPCSV), Sweet potato
virus G (SPVG) and Sweet
potato mild mottle virus
(SPMMV). a At 15 weeks
post inoculation, transgenic
plants T7 and T17 displayed
a milder level of disease
than transgenic plants T1,
T2, T3, and T4.
b Comparison of virus-
challenged non-transgenic
and transgenic plants
12 weeks post inoculation.
c Symptom reactions on
leaves of challenged non-
transgenic control plants
showing severe mosaic, leaf
strap, vein clearing,
crinkling and leaf curl.
d Asymptomatic upper leaf
(left) and symptomatic
lower leaf showing mild
mottle (right) of challenged
transgenic plants
384 Transgenic Res (2014) 23:377–388
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Results of the PCR (Fig. 1) and Southern blot
(Fig. 2) analyses indicated that the transgene construct
consisting of the partial CP segments of SPFMV,
SPCSV, SPVG and SPMMV was integrated into the
sweet potato genome. Southern blot analysis of six
transgenic plants showed one insertion loci with the
same pattern of hybridization. This suggested that all
six transgenic plants are not the result of independent
transformation events but are more likely to be clones
of one transgenic event.
Transgenic sweet potato plants were evaluated for
virus resistance by graft-inoculation with SPFMV,
SPCSV, SPVG and SPMMV. Sweet potato is a
vegetatively propagated plant and herbaceous cuttings
of the six transgenic sweet potato plants were used in
the evaluation. Following inoculation, SPFMV and
SPVG were detected in all transgenic plants (Table 1).
Therefore, none of the transgenic plants exhibited
immunity to virus infection, however, they displayed
delayed and milder symptoms of chlorosis and mottle
of lower leaves when compared to the untransformed
control plants (Fig. 3). This phenotype could be of
value if economic losses that result from severe
symptom expression are reduced.
It was expected that all transgenic plants, being
derived from the same transformation event, would
produce the same host reaction when challenged with
viruses. However, a higher degree of resistance was
displayed by transgenic plants T7 and T17 compared
to transgenic plants T1, T2, T3 and T4 (Table 1;
Fig. 3a). The latter plants proved more susceptible to
virus infection by exhibiting a more severe disease
reaction than transgenic plants T7 and T17. In
addition, weak positive reactions in the NCM-ELISA
for T7 and T17 indicated a low level of virus
accumulation in both the transgenic and indicator
plants. This variation can be attributed to the presence
of lower virus titer levels in scions used in the graft
inoculation of transgenic plants. An initial lower
concentration of virus levels in scions may result in a
lower concentration of virus in infected transgenic
plants which can manifest as slower and less severe
disease development. Other features such as a differ-
ential production of siRNA could also explain a
differential reaction to virus infection. Quantitative
ELISA and real-time RT-PCR may be used to provide
a better understanding of the virus titer associated with
varying levels of resistance.
Sweet potato chlorotic stunt virus and SPMMV
were not detected by NCM-ELISA in graft-inocu-
lated transgenic plants. This indicated that graft-
inoculation was not successful in the transfer of
these two viruses to transgenic and non-transgenic
plants. The dynamics of disease development
through graft inoculation might have favoured the
proliferation of SPFMV and SPVG over SPCSV
and SPMMV. Repeat grafting procedures should
focus on the inoculation of each virus singly rather
than simultaneous inoculation of all four viruses. In
this regard, insect inoculation using aphids (SPFMV
and SPVG) and whiteflies (SPCSV and SPMMV)
could provide a more efficient means of virus
transmission.
Previous studies have shown that resistance can be
overcome by graft-inoculations even if the sweet
potato genotype shows high-field resistance to virus
infection (Mwanga et al. 2002; Miano et al. 2008;
Okada and Saito 2008). SPFMV and SPVG are
transmitted by aphids and SPCSV and SPMMV by
whiteflies under natural conditions (Loebenstein et al.
2009). To fully determine the efficiency of the
transgene, further experimentation is needed in the
field where aphid and whitely inoculation will provide
a natural route to infection (Nyaboga et al. 2008). A
higher level of resistance could be obtained under field
Table 1 Symptom development and NCM-ELISA reactions
of plants graft-inoculated with Sweet potato feathery mottle
virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV),
Sweet potato virus G (SPVG) and Sweet potato mild mottle
virus (SPMMV)
Symptom reactions NCM-ELISA reactions
nSDMD-? ??
T1 5 - 5 -- 5 -
T2 5 - 5 -- 5 -
T3 5 - 5 -- 5 -
T4 5 - 5 -- 5 -
T7 5 -- 5 - 5 -
T17 5 -- 5 - 5 -
Control 10 10 -- - - 10
n, total number of graft-inoculated plants; S, plants with
development of severe mosaic, mottle, leaf strap, vein clearing,
crinkling and leaf curl 12 weeks post inoculation (wpi); D, plants
with delayed and attenuated symptom development of chlorosis and
mosaic 15 wpi; MD, plants with delayed and attenuated symptom
development of mosaic 15 wpi; -, negative ELISA reaction; ?,
weak positive ELISA reaction; ??, strong positive reaction
Transgenic Res (2014) 23:377–388 385
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conditions due to the lower and variable dose of
inoculum provided by vector transmission.
This study demonstrates that small segments of viral
genes can confer resistance when fused with a silencer
DNA. The usefulness of this strategy has been previ-
ously demonstrated to enhance the induction of gene
silencing. As previously described, the DNA silencer
was fused with a viral segment ensure the length of
transgene was long enough to induce gene silencing
(Pang et al. 1997;Jan1998; Jan et al. 2000b). Similarily,
Lin et al. (2011) showed that multiple resistance to
TSWV and ToLCTWV was obtained in tobacco and
tomato plants transformed with a single chimeric
transgene consisting of the partial N gene of TSWV
fused to the C2 gene segment of ToLCTWV. In this
study, the DNA silencer did not have an effect on the
resistance mechanism exhibited in transgenic sweet
potato plants. The approach used in this study was
through the linking of small gene segments of SPFMV,
SPVG SPCSV and SPMMV to the middle half of the N
gene of TSWV. Northern blot analysis of the resistant
plants for accumulation of transgene-derived siRNAs
would have provided evidence for PTGS.
Studies on the production of transgenic sweet
potato with resistance to SPFMV showed high levels
of protection in the greenhouse (Kreuze 2002). Once
these plants were subjected to field conditions, the
high levels of resistance to SPFMV broke down
following infection with SPCSV and the plants
succumb to severe SPVD (Karyeija et al. 2000;
Kreuze et al. 2008). Thus, infections by multiple
viruses is a severe impendent to the comprehensive
control of virus disease in sweet potato (Loebenstein
et al. 2009). The genetic engineering strategies
employed in this study show a novel approach of
addressing the problem of the multiple virus infec-
tions and synergism of sweet potato viruses.
Following field testing, this approach could be used
as the framework of controlling multiple virus
infection in other crops.
Acknowledgments We thank Dennis Gonsalves from US
Pacific Basin Agricultural Research Center, for providing the
plant expression silencing (pEPJ86-m/2N) and transformation
(pGA482G) vectors and Dr. P. Shanahan from the University of
KwaZulu-Natal for providing the sweet potato cultivar
‘Blesbok’. BJS was sponsored by the National Research
Foundation (NRF), South Africa for the entire duration of her
studies for which she is eternally grateful.
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